Oral treatment of inflammatory bowel disease

ABSTRACT

The present invention relates to treatment of an inflammatory bowel disease by simultaneous or successive parental and oral administration of a mammalian beta defensin.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/506,437, filed on Jul. 11, 2011 as well as priority under 35 U.S.C.§119 or 365 to European Application No. 11173351.5, filed Jul. 8, 2011.

The entire teachings of the above applications are incorporated hereinby reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

a) File name: Sequence Listing.txt; 3 KB in size.

BACKGROUND OF THE INVENTION Human Defensins

Among many other elements, key components of innate immunity are theantimicrobial peptides (AMPs) that individually show considerableselectivity, but collectively are able to rapidly kill a broad spectrumof bacteria, viruses and fungi. The biological significance of AMPs isemphasized by their ubiquitous distribution in nature and they areprobably produced by all multicellular organisms. In humans thepredominant AMPs are the defensins. The human defensins are smallcationic peptides that can be divided into α- and β-defensins based onthe topology of their three intramolecular cysteine disulphide bonds.The α-defensins can be further subdivided into those that were firstisolated from neutrophil granules (HNP1-4) and those that are expressedby Paneth cells in the crypts of the small intestine (HD5 and HD6). Theβ-defensins are mainly produced by epithelial cells in various tissuesand organs including the skin, trachea, gastrointestinal tract,urogenital system, kidneys, pancreas and mammary gland. The bestcharacterized members of the β-defensin family are hBD1-3. However,using various bioinformatics tools almost 40 open reading framesencoding putative β-defensin homologues have been annotated in the humangenome. Some of the human defensins are produced constitutively, whereasothers are induced by proinflammatory cytokines or exogenous microbialproducts.

It has become increasingly clear that the human defensins in addition totheir direct antimicrobial activity also have a wide range ofimmunomodulatory/alternative properties. These include the induction ofvarious chemokines and cytokines, chemotactic and apoptotic activities,induction of prostaglandin, histamine and leukotriene release,inhibition of complement, stimulation of dendritic cell maturationthrough toll-like receptor signalling and stimulation of pathogenclearance by neutrophils. Furthermore, the human defensins also play arole in wound healing, proliferation of epithelial and fibroblast cells,angiogenesis and vasculogenesis.

There is increasing evidence that the human defensins play an importantrole in many infectious and inflammatory diseases. Overexpression ofhuman defensins is often observed in inflamed and/or infected skin mostlikely because of local induction by microbial components or endogenousproinflammatory cytokines In psoriasis hBD2 and hBD3 are overabundantand in lesional epithelium of patients with acne vulgaris or superficialfolliculitis a significant upregulation of hBD2 has been observed. Onthe other hand, downregulation of hBD2 and hBD3 has been associated withatopic dermatitis. Ileal Crohn's disease has been associated withdeficient expression of HD5 and HD6 and in Crohn's disease in the colonexpression of hBD2-4 are downregulated.

Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) are defined by chronic, relapsingintestinal inflammation of obscure origin. IBD refers to two distinctdisorders, Crohn's disease and ulcerative colitis (UC). Both diseasesappear to result from the unrestrained activation of an inflammatoryresponse in the intestine. This inflammatory cascade is thought to beperpetuated through the actions of proinflammatory cytokines andselective activation of lymphocyte subsets. In patients with IBD, ulcersand inflammation of the inner lining of the intestines lead to symptomsof abdominal pain, diarrhea, and rectal bleeding. Ulcerative colitisoccurs in the large intestine, while in Crohn's, the disease can involvethe entire GI tract as well as the small and large intestines. For mostpatients, IBD is a chronic condition with symptoms lasting for months toyears. It is most common in young adults, but can occur at any age. Itis found worldwide, but is most common in industrialized countries suchas the United States, England, and northern Europe. It is especiallycommon in people of Jewish descent and has racial differences inincidence as well. Recent studies have identified variations in specificgenes, including ATG16L1, IL23R, IRGM, and NOD2, that influence the riskof developing Crohn disease. As many as 30 human genes have beenidentified which contribute to ulcerative colitis susceptibility.

The clinical symptoms of IBD are intermittent rectal bleeding, crampingabdominal pain, weight loss and diarrhea. Diagnosis of IBD is based onthe clinical symptoms, the use of a barium enema, but directvisualization (sigmoidoscopy or colonoscopy) is the most accurate test.Protracted IBD is a risk factor for colon cancer, and treatment of IBDcan involve medications and surgery.

Some patients with UC only have disease in the rectum (proctitis).Others with UC have disease limited to the rectum and the adjacent leftcolon (proctosigmoiditis). Yet others have UC of the entire colon(universal IBD). Symptoms of UC are generally more severe with moreextensive disease (larger portion of the colon involved with disease).

The prognosis for patients with disease limited to the rectum(proctitis) or UC limited to the end of the left colon(proctosigmoiditis) is better than that of full colon UC. Brief periodictreatments using oral medications or enemas may be sufficient. In thosewith more extensive disease, blood loss from the inflamed intestines canlead to anemia, and may require treatment with iron supplements or evenblood transfusions. Rarely, the colon can acutely dilate to a large sizewhen the inflammation becomes very severe. This condition is calledtoxic megacolon. Patients with toxic megacolon are extremely ill withfever, abdominal pain and distention, dehydration, and malnutrition.Unless the patient improves rapidly with medication, surgery is usuallynecessary to prevent colon rupture.

Crohn's disease can occur in all regions of the gastrointestinal tract.With this disease intestinal obstruction due to inflammation andfibrosis occurs in a large number of patients. Granulomas and fistulaformation are frequent complications of Crohn's disease. Diseaseprogression consequences include intravenous feeding, surgery andcolostomy.

IBD may be treated medicinally. The most commonly used medications totreat IBD are anti-inflammatory drugs such as the salicylates. Thesalicylate preparations have been effective in treating mild to moderatedisease. They can also decrease the frequency of disease flares when themedications are taken on a prolonged basis. Examples of salicylatesinclude sulfasalazine, azulfidine, olsalazine, and mesalamine. All ofthese medications are given orally in high doses for maximal therapeuticbenefit. These medicines are not without side effects. Azulfidine cancause upset stomach when taken in high doses, and rare cases of mildkidney inflammation have been reported with some salicylatepreparations.

Corticosteroids are more potent and faster-acting than salicylates inthe treatment of IBD, but potentially serious side effects limit the useof corticosteroids to patients with more severe disease. Side effects ofcorticosteroids usually occur with long term use. They include thinningof the bone and skin, increased susceptibility to infections, diabetes,hypertension, glaucoma, muscle wasting/weakness, rounding of the face,psychiatric disturbances, and, on rare occasions, destruction of hipjoints.

In IBD patients that do not respond to salicylates or corticosteroids,medications that suppress the immune system are used. Examples ofimmunosuppressants include azathioprine and 6-mercaptopurine.Immunosuppressants used in this situation help to control IBD and allowgradual reduction or elimination of corticosteroids. However,immunosuppressants render the patient immuno-compromised and susceptibleto many other diseases.

A well recognized model for studying IBD is the DSS colitis mouse model,as described in Kawada et al. “Insights from advances in research ofchemically induced experimental models of human inflammatory boweldisease”, World J. Gastroenterol., Vol. 13 (42), pp. 5581-5593 (2007);and Wirtz and Neurath “Mouse models of inflammatory bowel disease”,Advanced Drug Delivery Reviews, Vol. 59 (11), 1073-1083 (2007).

Treatment of inflammatory bowel disease (IBD) has traditionally beenaccomplished with administration of aminosalicylates, corticosteroids,thiopurines, methotrexate, and anti-tumor necrosis factor agents. Humanbeta defensins has long be thought to play a role in the occurrenceand/or treatment of IBD.

Clearly there is a great need for agents capable of preventing andtreating IBD.

WO 2007/081486 discloses the use of several human defensins in thetreatment of inflammatory bowel disease. The inventors suggested thatdefensins administered orally to Crohn's patients, in a formulation thatallow their release at proper locations in the intestinal lumen, wouldreduce the number of invading bacteria, re-establish a normal epithelialbarrier function and, thus, reduce the severity of the inflammatorydisease.

According to WO 2007/081486, the function of the defensins is todirectly target and kill bacteria in the lumen to prevent them frominvading the epithelial tissue. That is, the function of the defensinsis purely as an anti-infective compound.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention.

FIG. 1. Disease Activity Index (DAI) score (with body weight)progression during the study. Results are shown as the mean±the standarderror of the mean for n=10 animals per group. Significant differencesfrom control (vehicle) group values at a given date are shown as*P<0.05; **P<0.01 ***P<0.001 (Kruskal-Wallis Test for non-parametricdata).

FIG. 2. Histological score of proximal colon samples. Results are shownas the mean±the standard error of the mean for n=10 animals per group.Significant differences from control (vehicle) group values are shown as**P<0.01; ***P<0.001 (Kruskal-Wallis Test+post-test of Dunn fornon-parametric data).

FIG. 3. Histological score of distal colon samples. Results are shown asthe mean±the standard error of the mean for n=10 animals per group.Significant differences from control (vehicle) group values are shown as**P<0.01 (Kruskal-Wallis Test+post-test of Dunn for non-parametricdata).

FIG. 4. Clustal W (2.1) multiple sequence alignment of human, Rhesusmacaque, chimpanzee and orangutan beta defensin 2.

-   -   * indicates positions which have a single, fully conserved        residue.    -   : indicates that one of the following ‘strong’ groups is fully        conserved:        -   STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.    -   . indicates that one of the following ‘weaker’ groups is fully        conserved:        -   CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK,            VLIM, HFY.

FIG. 5. Clustal W (2.1) multiple sequence alignment of human defensin1-4.

-   -   * indicates positions which have a single, fully conserved        residue.    -   : indicates that one of the following ‘strong’ groups is fully        conserved:        -   STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.    -   . indicates that one of the following ‘weaker’ groups is fully        conserved:        -   CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK,            VLIM, HFY.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Definitions:

-   Defensin: The term “defensin” as used herein refers to polypeptides    recognized by a person skilled in the art as belonging to the    defensin class of antimicrobial peptides. To determine if a    polypeptide is a defensin according to the invention, the amino acid    sequence may be compared with the hidden markov model profiles (HMM    profiles) of the PFAM database by using the freely available HMMER    software package.

The PFAM defensin families include for example Defensin_(—)1 or“Mammalian defensin” (accession no. PF00323), and Defensin_(—)2 orDefensin_beta or “Beta Defensin” (accession no. PF00711).

The defensins of the invention belong to the beta defensin class. Thedefensins from the beta defensin class share common structural featuresknown to the person skilled in the art, such as the cysteine pattern.

Examples of defensins, according to the invention, include human betadefensin 1 (hBD1; see SEQ ID NO:1), human beta defensin 2 (hBD2; see SEQID NO:2), human beta defensin 3 (hBD3; see SEQ ID NO:3), human betadefensin 4 (hBD4; see SEQ ID NO:4), and mouse beta defensin 3 (mBD3; seeSEQ ID NO:6).

-   Identity: The relatedness between two amino acid sequences or    between two nucleotide sequences is described by the parameter    “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277; http://emboss.org), preferably version3.0.0 or later. The optional parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version ofBLOSUM62) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

-   Isolated polypeptide: The term “isolated variant” or “isolated    polypeptide” as used herein refers to a variant or a polypeptide    that is isolated from a source. In one aspect, the variant or    polypeptide is at least 1% pure, preferably at least 5% pure, more    preferably at least 10% pure, more preferably at least 20% pure,    more preferably at least 40% pure, more preferably at least 60%    pure, even more preferably at least 80% pure, and most preferably at    least 90% pure, as determined by RP-HPLC analysis.-   Substantially pure polypeptide: The term “substantially pure    polypeptide” denotes herein a polypeptide preparation that contains    at most 10%, preferably at most 8%, more preferably at most 6%, more    preferably at most 5%, more preferably at most 4%, more preferably    at most 3%, even more preferably at most 2%, most preferably at most    1%, and even most preferably at most 0.5% by weight of other    polypeptide material with which it is natively or recombinantly    associated. It is, therefore, preferred that the substantially pure    polypeptide is at least 92% pure, preferably at least 94% pure, more    preferably at least 95% pure, more preferably at least 96% pure,    more preferably at least 96% pure, more preferably at least 97%    pure, more preferably at least 98% pure, even more preferably at    least 99%, most preferably at least 99.5% pure, and even most    preferably 100% pure by weight of the total polypeptide material    present in the preparation. The polypeptides of the present    invention are preferably in a substantially pure form. This can be    accomplished, for example, by preparing the polypeptide by    well-known recombinant methods or by classical purification methods    or by chemical synthesis.

Remission: The term “Remission” denotes herein periods with diseasecontrol versus active disease which are often referred to as “attack”.

Relapse: The term “relapse” denotes herein re-occurence of the symptomsof IBD. The most common early symptoms of IBD are chronic diarrhea(which is sometimes bloody), cramping abdominal pain, fever, loss ofappetite, and weight loss. Symptoms may continue for days or weeks andmay resolve without treatment. IBD relapses at irregular intervalsthroughout the lifespan of a subject. Relapse can be mild or severe,brief or prolonged. Severe relapses can lead to intense pain,dehydration, and blood loss.

Inflammatory bowel disease (IBD): inflammatory bowel disease (IBD) is agroup of inflammatory conditions of the colon and small intestine. Themajor types of IBD are Crohn's disease and ulcerative colitis (UC).

Accounting for far fewer cases are other forms of IBD, which are notalways classified as typical IBD:

-   -   Collagenous colitis    -   Lymphocytic colitis

Ischaemic colitis

-   -   Diversion colitis    -   Behçet's disease    -   Indeterminate colitis

The main difference between Crohn's disease and UC is the location andnature of the inflammatory changes. Crohn's can affect any part of thegastrointestinal tract, from mouth to anus (skip lesions), although amajority of the cases start in the terminal ileum. Ulcerative colitis,in contrast, is restricted to the colon and the rectum.

Treatment: The terms “treatment” and “treating” as used herein refer tothe management and care of a patient for the purpose of combating acondition, disease or disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of the active compound for the purposeof: alleviating or relieving symptoms or complications; delaying theprogression of the condition, disease or disorder; curing or eliminatingthe condition, disease or disorder; and/or preventing the condition,disease or disorder, wherein “preventing” or “prevention” is to beunderstood to refer to the management and care of a patient for thepurpose of hindering, reducing or delaying the development of thecondition, disease or disorder, and includes the administration of theactive compounds to prevent or reduce the risk of the onset of symptomsor complications. The patient to be treated is preferably a mammal, inparticular a human being. The patients to be treated according to thepresent invention can be of various ages.

Mammal Beta Defensins

The present invention relates to pharmaceutical uses of mammal betadefensins, such as mouse, monkey or human beta defensins, morepreferably Hominidae beta defensins, more preferably human betadefensin. in the treatment of inflammatory bowel diseases, such asulcerative colitis and/or Crohns disease.

In an embodiment, the mammal beta defensins of the invention have adegree of identity of at least 80%, preferably at least 85%, morepreferably at least 90%, and most preferably at least 95% to any of theamino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5 and/or SEQ ID NO:6. In a preferred embodiment, themammal beta defensins of the invention have a degree of identity of atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% to any of the amino acid sequences of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3 and/or SEQ ID NO:4. In a morepreferred embodiment, the mammal beta defensins of the invention consistof human beta defensin 1 (SEQ ID NO:1), human beta defensin 2 (SEQ IDNO:2), human beta defensin 3 (SEQ ID NO:3), human beta defensin 4 (SEQID NO:4), a variant of human beta defensin 4 (SEQ ID NO:5) and/or mousebeta defensin 3 (SEQ ID NO:6). In an even more preferred embodiment, themammal beta defensins of the invention consist of human beta defensin 1(SEQ ID NO:1), human beta defensin 2 (SEQ ID NO:2), human beta defensin3 (SEQ ID NO:3) and/or human beta defensin 4 (SEQ ID NO:4).

In a preferred embodiment, the mammal beta defensins of the inventionhave a degree of identity of at least 80%, preferably at least 85%, morepreferably at least 90%, and most preferably at least 95% to the aminoacid sequence of SEQ ID NO:2. In a preferred embodiment, the mammal betadefensins of the invention consist of human beta defensin 2 (SEQ IDNO:2).

In yet another embodiment, the mammal beta defensins of the inventionconsist of human beta defensins and/or mouse beta defensins, andfunctionally equivalent variants thereof. Preferably, the mammal betadefensins consist of human beta defensin 1, human beta defensin 2, humanbeta defensin 3, human beta defensin 4, Chimpanzee beta defensin 2,Macaque beta defensin 2, and mouse beta defensin 3, and functionallyequivalent variants thereof. More preferably, the mammal beta defensinsof the invention consist of human beta defensin 2, and functionallyequivalent variants thereof.

The mammal beta defensins of the invention are also referred to ascompounds of the preferred embodiments.

In the context of the present invention, a “functionally equivalentvariant” of a mammal (e.g. human) beta defensin is a modified mammal(e.g. human) beta defensin exhibiting approx. the same effect on aninflammatory bowel disease as the parent mammal (e.g. human) betadefensin. Preferably, it also exhibits approx. the same effect on theactivity of pro-inflammatory cytokines and chemokines and/oranti-inflammatory cytokine as the mammal (e.g. human) beta defensin. Inone embodiment the effect on the activity of pro-inflammatory andanti-inflammatory cytokines is measured as described in WO 2010/007166in Example 4 and 6. The effect may be measured in a human cell selectedfrom the group consisting of human PBMCs, human CD14+ monocyte-deriveddendritic cells, a human monocyte cell line, and human immaturedendritic cells, preferably human PBMC.

According to the invention, a functionally equivalent variant of amammal (e.g. human) beta defensin may comprise 1-5 amino acidmodifications, preferably 1-4 amino acid modifications, more preferably1-3 amino acid modifications, most preferably 1-2 amino acidmodification(s), and in particular one amino acid modification, ascompared to the mammal (e.g. human) beta defensin amino acid sequence.Preferably compared to human beta defensin 2, having SEQ ID NO 2.

The term “modification” means herein any chemical modification of amammal (e.g. human) beta defensin. The modification(s) can besubstitution(s), deletion(s) and/or insertions(s) of the amino acid(s)as well as replacement(s) of amino acid side chain(s); or use ofunnatural amino acids with similar characteristics in the amino acidsequence. In particular the modification(s) can be amidations, such asamidation of the C-terminus.

Preferably, amino acid modifications are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the polypeptide;single deletions; small amino- or carboxyl-terminal extensions; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tag, an antigenic epitope ora binding domain. In one embodiment the small extension, such as apoly-histidine tag, an antigenic epitope or a binding domain is attachedto the mammalian (e.g. human) beta defensin through a small linkerpeptide of up to about 20-25 residues and said linker may contain arestriction enzyme cleavage site. The Clustal W alignment in FIG. 4 orFIG. 5 can be used to predict which amino acid residues can besubstituted without substantially affecting the biological acitivity ofthe protein. The sequences were aligned using Clustal W 2.1(www.genome.jp/tools/clustalw/) and the following settings: Gap OpenPenalty:10, Gap Extension Penalty: 0.05, Weight Transition: NO,Hydrophilic Residues for Proteins:GPSNDQE, Hydrophilic Gaps: YES,

-   Weight Matrix: BLOSUM (for PROTEIN).

Substutions within the following group (Clustal W, ‘strong’ conservationgroup) are to be regarded as conservative substitutions within themeaning of the present invention

-   -   STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Substutions within the following group (Clustal W, ‘weak’ conservationgroup) are to be regarded as semi-conservative substitutions within themeaning of the present invention

-   -   CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM,        HFY.

Examples of conservative substitutions are substitutions made within thegroup of basic amino acids (arginine, lysine and histidine), acidicamino acids (glutamic acid and aspartic acid), polar amino acids(glutamine and asparagine), hydrophobic amino acids (leucine, isoleucineand valine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine, threonineand methionine). Amino acid substitutions which do not generally alterspecific activity are known in the art and are described, for example,by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press,New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in a mammal beta defensin can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,activity against an inflammatory bowel disease and/or suppresion ofTNF-alpha activity) to identify amino acid residues that are critical tothe activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to mammal beta defensins (see Clustal W alignment in FIG. 4 andFIG. 5).

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

An N-terminal extension of the polypeptides of the invention maysuitably consist of from 1 to 50 amino acids, preferably 2-20 aminoacids, especially 3-15 amino acids. In one embodiment N-terminal peptideextension does not contain an Arg (R). In another embodiment theN-terminal extension comprises a kex2 or kex2-like cleavage site as willbe defined further below. In a preferred embodiment the N-terminalextension is a peptide, comprising at least two Glu (E) and/or Asp (D)amino acid residues, such as an N-terminal extension comprising one ofthe following sequences: EAE, EE, DE and DD.

When the result of a given substitution cannot be predicted withcertainty, the derivatives may be readily assayed according to themethods described herein above to determine the presence or absence ofbiological activity.

Methods and Uses

Human beta defensin 2 was found to significantly reduce the severity ofdisease parameters in a 10-Day Dextran Sodium Sulphate (DSS)-inducedcolitis model in the mouse; thus showing potent activity as a medicamentfor treatment of inflammatory bowel diseases, such as ulcerative colitisand Crohn's disease.

The present invention therefore provides methods of prophylactictreatment for an inflammatory bowel diseases or as a method of keeping asubject suffering from IBD in remission, which treatment comprisesadministering orally to a subject in need of such treatment an effectiveamount of a mammal beta defensin, such as human beta defensin 2, e.g.,in the form of a pharmaceutical composition.

In one embodiment the invention relates to prolonging this period ofremission after oral administration of a mammalian beta defensin orreducing the occurrence of relapse in a subject suffering from IBD.

Inflammatory bowel disease according to this invention relates toCrohn's Disease and ulcerative colitis. In a preferred embodiment thestage of Crohn's Disease to be treated using oral administration of amammalian beta defensin is remission and/or mild active Crohn's Disease.In another preferred embodiment the stage of ulcerative colitis to betreated using oral administration of a mammalian beta defensin isremission stage of ulcerative colitis (e.g. pancolitis/remission,left-sided colitis/remission and therapy-refractory disease inremission).

In one embodiment of the present invention oral administration ofmammalian beta defensions may be used for reducing neutrophil invasionin the epithelial lining of the colon. This reduction may be located toeither the proximal or the distal colon.

In another embodiment of the present invention oral administration ofmammalian beta defensin may be used for treatment of inflammation in thecolon.

In yet another embodiment of the present invention oral administrationof a mammalian beta defensin may be used for modulating cytokineproduction at the epithelial lining in the gastrointestinal tract.

The modulation is preferably a downregulation of the activity of atleast one cytokine selected from the group consisting of thepro-inflammatory cytokines and chemokines Preferably the modulation is adownregulation of the activity of at least one cytokine selected fromthe group consisting of the pro-inflammatory IL-23, IL-β and TNFα,and/or an upregulation of the activity of the anti-inflammatory cytokineIL-10.

In another embodiment the present invention provides methods of treatinginflammatory bowel diseases, which treatment comprises administeringsimultaneous or successive parenteral and oral administration of aneffective amount of a mammalian beta defensin in the form of apharmaceutical composition. During an active breakout (“attack”) ofinflammatory bowel disease the subject suffering from said IBD may inone embodiment be subjected to parenteral administration of a mammalianbeta defensin, such as human beta defensin 2, combined with or followedby oral administration of said mammalian beta defensin during theperiods of remission.

In an embodiment, the method of treatment with hBD2 results in asignificant inhibition of the disease activity index (DAI) in an in vivomodel of IBD when administered twice daily, at 3 dose levels by the oralroute and hBD2 also demonstrated a very significant anti-inflammatoryactivity in the same model.

Mammal beta defensins can be employed therapeutically in compositionsformulated for administration by any conventional route. In oneembodiment of the present invention mammalian beta defensins areadministered orally.

In another embodiment of the present invention mammalian beta defensinsare administered by simultaneous or successive oral and parenteraladministration.

Oral administration is normally for enteral drug delivery, wherein theagent is delivered through the enteral mucosa.

Parenteral administration is any administration route not being theoral/enteral route whereby the medicament avoids first-pass degradationin the liver. Accordingly, parenteral administration includes anyinjections and infusions, for example bolus injection or continuousinfusion, such as intravenous administration, intramuscularadministration, subcutaneous administration.

The subcutaneous and intramuscular forms of parenteral administrationare generally preferred.

Within yet other embodiments, compositions, of preferred embodiments maybe formulized as a lyophilizate, utilizing appropriate excipients thatprovide stability as a lyophilizate, and subsequent to rehydration.

Pharmaceutical compositions containing a mammal beta defensin, such as ahuman beta defensin, can be manufactured according to conventionalmethods, e.g., by mixing, granulating, coating, dissolving orlyophilizing processes.

Pharmaceutical compositions of preferred embodiments comprise a mammalbeta defensin, such as a human beta defensin, and a pharmaceuticallyacceptable carrier and/or diluent.

Pharmaceutically acceptable carriers and/or diluents are familiar tothose skilled in the art. For compositions formulated as liquidsolutions, acceptable carriers and/or diluents include saline andsterile water, and may optionally include antioxidants, buffers,bacteriostats, and other common additives.

Parenteral administration may be in the form of injections and infusionsand said formulations may take such forms as suspensions, solutions, oremulsions in oily or aqueous vehicles. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution for constitution beforeuse with a suitable vehicle, e.g., sterile, pyrogen-free water. Theformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules, vials, pre-filled syringes, infusion bags,or can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid excipient, for example, water,for injections, immediately prior to use.

Examples of oily or non-aqueous carriers, diluents, solvents or vehiclesinclude propylene glycol, polyethylene glycol, vegetable oils, andinjectable organic esters, and may contain formulatory agents such aspreserving, wetting, emulsifying or suspending, stabilizing and/ordispersing agents.

The compound of the present invention may be formulated in a widevariety of formulations for oral administration. Solid form preparationsmay include powders, tablets, drops, capsules, cachets, lozenges, anddispersible granules. Other forms suitable for oral administration mayinclude liquid form preparations including emulsions, syrups, elixirs,aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice,chewing gum, or solid form preparations which are intended to beconverted shortly before use to liquid form preparations, such assolutions, suspensions, and emulsions. In powders, the carrier is afinely divided solid which is a mixture with the finely divided activecomponent. In tablets, the active component is mixed with the carrierhaving the necessary binding capacity in suitable proportions andcompacted in the shape and size desired. Suitable carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.

Drops according to the present invention may comprise sterile ornon-sterile aqueous or oil solutions or suspensions, and may be preparedby dissolving the active ingredient in a suitable aqueous solution,optionally including a bactericidal and/or fungicidal agent and/or anyother suitable preservative, and optionally including a surface activeagent. Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Emulsions may be prepared in solutions in aqueous propylene glycolsolutions or may contain emulsifying agents such as lecithin, sorbitanmonooleate, or acacia. Aqueous solutions can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizing and thickening agents. Aqueous suspensions can be preparedby dispersing the finely divided active component in water with viscousmaterial, such as natural or synthetic gums, resins, methylcellulose,sodium carboxymethylcellulose, and other well known suspending agents.

The formulation can contain (in addition to a mammal beta defensin, andother optional active ingredients) carriers, fillers, disintegrators,flow conditioners, sugars and sweeteners, fragrances, preservatives,stabilizers, wetting agents, emulsifiers, solubilizers, salts forregulating osmotic pressure, buffers, diluents, dispersing andsurface-active agents, binders, lubricants, and/or other pharmaceuticalexcipients as are known in the art.

One skilled in this art may further formulate mammal beta defensins inan appropriate manner, and in accordance with accepted practices, suchas those described in Remington's Pharmaceutical Sciences, Gennaro, Ed.,Mack Publishing Co., Easton, Pa. 1990.

A mammal beta defensin, such as a human beta defensin, can be usedalone, or in combination therapies with one, two, or more otherpharmaceutical compounds or drug substances, and/or with one or morepharmaceutically acceptable excipient(s).

In Vitro Synthesis

Mammal beta defensins may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example automated synthesizers by AppliedBiosystems Inc., Beckman, etc. By using synthesizers, naturallyoccurring amino acids may be substituted with unnatural amino acids,particularly D-isomers (or D-forms) e.g. D-alanine and D-isoleucine,diastereoisomers, side chains having different lengths orfunctionalities, and the like. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

Chemical linking may be provided to various peptides or proteinscomprising convenient functionalities for bonding, such as amino groupsfor amide or substituted amine formation, e.g. reductive amination,thiol groups for thioether or disulfide formation, carboxyl groups foramide formation, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

Mammal beta defensins, or functional equivalents thereof, may also beisolated and purified in accordance with conventional methods ofrecombinant synthesis. Recombinant synthesis may be performed usingappropriate expression vectors and a eukaryotic expression system. Asolution may be prepared of the expression host and the media and thedefensins present purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique. Methods for recombinant expression of human beta defensin 2in E. coli are disclosed in WO 2010/007166 (Novozymes).

Dosages

A mammal beta defensin, such as a human beta defensin, is preferablyemployed in pharmaceutical compositions in an amount which is effectiveto treat an inflammatory bowel disease, preferably with acceptabletoxicity to the patient. For such treatment, the appropriate dosagewill, of course, vary depending upon, for example, the chemical natureand the pharmacokinetic data of a compound of the present inventionused, the individual host, the mode of administration and the nature andseverity of the conditions being treated. However, in general, forsatisfactory results in larger mammals, for example humans, an indicateddaily dosage is preferably from about 0.0001 mg/kg body weight to about10 mg/kg body weight, more preferably from about 0.001 mg/kg body weightto about 10 mg/kg body weight; such as 0.005 mg/kg body weight to 5mg/kg body weight, more preferably from about 0.01 mg/kg body weight toabout 10 mg/kg body weight, preferably from about 0.1 mg/kg body weightto about 10 mg/kg body weight, for example, administered in divideddoses up to one, two, three, or four times a day. The compounds ofpreferred embodiments can be administered to larger mammals, for examplehumans, by similar modes of administration at similar dosages thanconventionally used.

In one embodiment the daily dosage is preferably from 0.0001-10 mg/kgbodyweight, more preferably 0.001-10 mg/kg body weight, more preferably0.005-5 mg/kg body weight.

In certain embodiments, the pharmaceutical compositions of preferredembodiments can include a mammal beta defensin, such as a human betadefensin, in an amount of about 0.5 mg or less to about 1500 mg or moreper unit dosage form depending upon the route of administration,preferably from about 0.5, 0.6, 0.7, 0.8, or 0.9 mg to about 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg, and morepreferably from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg toabout 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg.In certain embodiments, however, lower or higher dosages than thosementioned above may be preferred. Appropriate concentrations and dosagescan be readily determined by one skilled in the art.

In one embodiment, the mammalian beta defensin is administered at leastonce daily, such as at least twice daily, for example at least 3 timesdaily, such as at least 4 times daily.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1

10-Day Dextran Sodium Sulphate (DSS)-Induced Colitis Model in the Mouse

The aim of the following study was to determine the anti-inflammatoryactivity of human beta defensin 2 in an acute (10-days) model ofinflammatory bowel disease (colitis) induced by oral dextran sodiumsulphate (DSS) administration in the mouse.

The DSS colitis mouse model is a well recognized model for studyinginflammatory bowel disease, as described in Kawada et al. “Insights fromadvances in research of chemically induced experimental models of humaninflammatory bowel disease”, World J. Gastroenterol., Vol. 13 (42), pp.5581-5593 (2007); and Wirtz and Neurath “Mouse models of inflammatorybowel disease”, Advanced Drug Delivery Reviews, Vol. 59 (11), 1073-1083(2007).

Materials

Test Items

Human beta defensin 2 (hBD2); Methylprednisolone 21-hemisuccinate(“prednisolone”), PBS buffer (GIBCO).

Experimental Animals

Male C57BL/6 mice (Harlan Interfauna Iberica, Barcelona, Spain) wereused in the study, as this is a species and sex that has beendemonstrated to develop significant inflammation of the colon whenadministered a 2% solution of DSS in the drinking water over a period of10 days.

Identification

Animals were identified by number and letter codes on their tails.Additionally, each cage was identified by a colour-coded card indicatingthe number and sex of the animals, the test item code or name, doselevel, administration route, treatment period, group number, study codeand study director's name.

Weight

The average body weight of the animals on the day of start of the studywas 22.4±0.16 g.

Acclimatization

Minimum of 7 days prior to the start of the study, under the sameconditions as those of the main study.

Housing

On arrival, the animals were separated and housed at random inpolycarbonate cages (E-Type, Charles River, 255×405×197 mm) withstainless steel lids. Animals were housed in groups of five animals percage according to their sex, in animal rooms with controlled temperature(22±2° C.), lighting (12/12 hours light/darkness), air pressure, numberof air renovations and relative humidity (30-70%). The cages all hadsawdust (Lignocel 3-4; Harlan Interfauna Iberica, Spain) on the floor aslitter. All mice had free access to a dry, pelleted standard rodent diet(Teklad Global 2014; Harlan Interfauna Iberica, Spain). Water wasprovided ad libitum in bottles. Tap water supply to the animal rooms isperiodically analysed to check its composition and to detect possiblecontaminants (chemical and microbiological).

Equipment and Materials

Equipment:

Animal balance Sartorius Mod. BP 2100

Surgical dissection equipment

Eppendorf 5415 C centrifuge

Nikon Eclipse E600FN microscope

Hook & Tucker instruments rotamixer

IKA Ultra Turrax Homogeniser

Sartorius Mod. BP 221S analytical balance

Materials and Reagents:

Sterile disposable syringes (1 ml)

Sterile Butterfly 25 G infusion set

Anaesthetic (Ketamine/Xylazine)

Topical Anaesthetic cream (EMLA, Astra Zeneca)

Dextran Sodium Sulphate 30.000-50.000 Da (MP Biomedicals)

Phosphate Buffered Saline (PBS; Sigma)

Neutral Buffered Formalin (VWR)

Experimental Protocol

Study design: animals were divided into 5 experimental groups. Eachgroup consisted of 10 males: Group A: Treated with Control vehicle (PBS)p.o. twice daily. Group B: Treated with methylprednisolone (1 mg/kg p.o.twice daily). Group C: Treated with hBD2 (0.05 mg/kg p.o. twice daily).Group D: Treated with hBD2 (0.5 mg/kg p.o. daily). Group E: Treated withhBD2 (5 mg/kg p.o. daily). Animal allocation to all experimental groupswas done in a randomized manner. A maximum of 5 mice were housed in eachcage (as per Directive 86/609/EEC). All animals were weighed on theirarrival at the laboratory and prior to the administration of the testitems.

Administration of the Test Substance

The control vehicle and hBD2 were administered orally with the use of asterile cannula in a dosing volume of 10 ml/kg body weith. The animalsof all groups recieved 2 doses daily (at 9.00 h and 17.00 h) of thecorresponding test item (test, reference or vehicle) for 10 consecutivedays (experimental days 1-10).

Experimental Procedure

Induction of Colitis

Colitis was induced in mice by supplementing their drinking water withDSS 2% for 7 days. On Day 1 all mice were weighed and marked accordingto their experimental groups. The drinking bottle of each cage wasfilled with the DSS solution, making sure all bottle lids were mountedproperly and that none were congested. On Day 3 any remaining solutionin the bottles was emptied and refilled with fresh DSS solution. Thisprocedure was repeated again on Day 5. On Day 8 any remaining solutionwas discarded and replaced with autoclaved water. Animals weresacrificed 2 days later on Day 10.

Clinical Assessment (Disease Activity Index)

Daily clinical assessment of DSS-treated animals was carried out, withthe calculation of a validated clinical Disease Activity Index (DAI)ranging from 0 to 4 according to the following parameters: stoolconsistency, presence or absence of rectal bleeding and weight loss:

Description Score Change in Body Weight:  <1% 0   1-5% 1  >5-10%2 >10-15% 3 >15% 4 Rectal Bleeding: Negative 0 Positive 4 StoolConsistency: Normal 0 Loose Stools 2 Diarrhoea 4

Bodyweight loss was calculated as the percent difference between theoriginal bodyweight (Day 1) and the actual bodyweight on eachexperimental day (2-10). The appearance of diarrhoea is defined asmucus/faecal material adherent to anal fur. Rectal bleeding is definedas diarrhoea containing visible blood/mucus or gross rectal bleeding.The maximum score of the DAI each day is 12.

Euthanasia and Collection of Colon Samples

On day 10, two hours after the last administration of control vehicle,hBD2 or prednisolone, the animals were killed by an overdose ofanaesthetic. Their colons were removed and their length and weightmeasured after exclusion of the caecum. Two sections (proximal anddistal) of colon were taken from each animal and preserved in neutralbuffered formalin for subsequent histological analysis (haematoxylin andeosin staining) according to the following scoring system:

Description Score: No changes observed (0). Minimal scattered mucosalinflammatory cell infiltrates, with or without minimal epithelialhyperplasia (1). Mild scattered to diffuse inflammatory cellinfiltrates, sometimes extending into the submucosa and associated witherosions, with minimal to mild epithelial hyperplasia and minimal tomild mucin depletion from goblet cells (2). Mild to moderateinflammatory cell infiltrates that were sometimes transmural, oftenassociated with ulceration, with moderate epithelial hyperplasia andmucin depletion (3). Marked inflammatory cell infiltrates that wereoften transmural and associated with ulceration, with marked epithelialhyperplasia and mucin depletion (4). Marked transmural inflammation withsevere ulceration and loss of intestinal glands (5).

Histological Evaluation

Two sections (proximal and distal) of colon were taken from each animal,processed for histological analysis (haematoxylin and eosin staining)and scored by a blind observer according to the histological scoringsystem described above.

Statistical Analysis

The statistical significance of the results was evaluated using thestatistics program GraphPad Instat 3. Results are expressed as themean±standard error of the mean, where (n) is the number of animals. Thedifference between groups for body weight, colon weight and colon lengthwas evaluated by Analysis of Variance (ANOVA) for unpaired data pluspost-test Dunnett to allow for multiple comparisons. The differencebetween groups for disease activity index and histological score wasevaluated by Kruskal-Wallis test for unpaired data+post-test of Dunn formultiple comparisons. A value of P<0.05 was taken as significant. In thefigures and tables, significant differences versus the correspondingcontrol (vehicle) group are denoted as: *P<0.05, **P<0.01, ***P<0.001.

Results

In the present study, treatment with the test item NZ39000 (hBD2)resulted in a significant inhibition of the disease activity index (DAI)when administered daily, at three dose levels (0.05, 0.5, 5 mg/kg) viathe oral route (FIG. 1 and tables below). A significant inhibition wasobserved after treatment with both the reference item (prednisolone) andwith hBD2 with the following significant differences:

Days or treatments not mentioned was not significant different.

hBD2 0.05 mg/kg Statistical significance Day NZ39000 DAI Vehicle DAI vs.vehicle 6  1.2 ± 0.59  4.4 ± 0.72 P < 0.01  7  1.9 ± 0.66  5.7 ± 0.92 P< 0.05  8  4.5 ± 0.86  9.2 ± 0.25 P < 0.001 9  5.6 ± 0.54 10.2 ± 0.57 P< 0.001 10   8 ± 0.73 11.6 ± 0.4  P < 0.05 

hBD2 0.5 mg/kg Statistical significance Day NZ39000 DAI Vehicle DAI vs.vehicle 6  1.7 ± 0.67  4.4 ± 0.72 P < 0.05 9  7.1 ± 0.61 10.2 ± 0.57 P <0.05 10 7.3 ± 0.7 11.6 ± 0.4  P < 0.01

hBD2 5 mg/kg Statistical significance Day NZ39000 DAI Vehicle DAI vs.vehicle 3  0.5 ± 0.27 2.6 ± 0.7 P < 0.05  4  0.6 ± 0.27  2.6 ± 0.75 P <0.05  5  1.8 ± 0.76  4.4 ± 0.72 P < 0.05  6 1.4 ± 0.6  4.4 ± 0.72 P <0.05  7  2.1 ± 0.96  5.7 ± 0.92 P < 0.05  8  5.4 ± 0.87  9.2 ± 0.25 P <0.01  9  5.2 ± 0.76 10.2 ± 0.57 P < 0.001 10  5.6 ± 0.48 11.6 ± 0.4  P <0.001

Prednisolone 1 mg/kg Statistical significance Day Pred. DAI Vehicle DAIvs. vehicle 6 7 ± 0.33 11.6 ± 0.4 P < 0.01

A significant reduction was also observed for the both proximal anddistal colon (FIGS. 2 and 3).

Conclusions

The results demonstrate that orally administered hBD2 at the testeddoses significantly reduces the increase in Disease Activity Indexinduced by DSS administration at day 6, 7, 8, 9, and 10 for 0.05 mg/kg,at day 6, 9 and 10 for 0.5 mg/kg and at day 3, 4, 5, 6, 7, 8, 9 and 10at 5 mg/kg. Similarly to the results obtained with the Disease ActivityIndex, histological analysis of the proximal and distal colons of eachanimal on day 10 revealed a very significant reduction of histologicaldamage score by orally administered hBD2. The effect was more pronouncedin the proximal than the distal colons. In the present study, treatmentwith the test item hBD2 resulted in a significant inhibition of thedisease activity index (DAI) when administered daily, at 3 dose levelsby the oral route. The reference item (Prednisolone) is known to have asignificant appetite suppressant effect in mice (and it was administeredtwice each day), which can in turn result in a significant effect overanimal body weight unrelated to the severity of the disease. To takethis effect into consideration, an additional DAI score was also carriedout without the change in body weight effect, only stool consistency andrectal bleeding were used as measurable parameters. In this case thetest item hBD2 also demonstrated a very significant anti-inflammatoryactivity, as did the reference item prednisolone. Administration ofdextran sodium sulphate resulted in a significant inflammation andinjury of the animals’ colonic tissue, as evidenced after histologicalexamination. This injury was more pronounced in the distal section ofthe colon when compared with the proximal section. Treatment with thetest item hBD2 twice daily, at 3 dose levels, resulted in a significantreduction of this histological damage in the proximal colon. Similarly,treatment with the test item hBD2 twice daily, at the intermediate andhigh dose levels, also resulted in a significant reduction of thehistological damage in the distal colon. Treatment with prednisolone ata dose of 1 mg/kg p.o. twice daily (Group B) also significantly reducedthe histological injury induced by DSS in the proximal and the distalcolon. The results also show a significant increase in body weight inthe animals treated with the high dose of the test item on day 10(P<0.05) when compared to the control group. This result would suggestan improvement in the animals' general health status in the test itemgroups mentioned leading to less loss of body weight (for example byeating more, less diarrhea, etc). The animals treated with prednisolone(group B) show a significant decrease in body weight from day 2 untilthe end of the study. As mentioned previously, this decrease in bodyweight cannot be taken as an indication of general health status of theanimals in this particular case, as prednisolone is known to have asignificant inhibitory effect over appetite in mice, which would explainthe decrease in body weight. The results obtained in the present studydemonstrate an anti-inflammatory activity of orally administered hBD2 inthe model of DSS colitis induced in the mouse after a 10-day treatmentperiod.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of treatment of an inflammatory boweldisease, the method comprising simultaneous or successive oral andparenteral administration of a therapeutically effective amount of oneor more mammalian beta defensins to a subject in need thereof.
 2. Themethod of claim 1, wherein the one or more defensins are administeredorally after at least one occasion of parenteral administration of oneor more defensins.
 3. The method of claim 1, wherein the one or moredefensins are administered orally together with at least one parenteraladministration of a dosage of one or more defensins.
 4. A method ofkeeping a subject suffering from an inflammatory bowel disease inremission or prolonging remission, the method comprising oraladministration of a therapeutically effective amount of one or moremammalian beta defensins to the subject.
 5. The method of claim 1,wherein the subject suffers from Crohn's disease.
 6. The method of claim1, wherein the subject suffers from mild active disease or moderately toseverely active disease.
 7. The method of claim 1, wherein the subjectsuffers from ulcerative collitis.
 8. The method of claim 7, wherein thecolitis is a pancolitis or left sided colitis.
 9. The method of claim 1,wherein the subject is human and the one or more mammalian betadefensins are one or more human beta defensins.
 10. The method of claim1, wherein the one or more mammalian beta defensins comprise an aminoacid sequence having at least 80% sequence identity to any one of theamino acid sequences selected from the group consisting of SEQ ID NO 1,SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO
 4. 11. The method of claim 1,wherein the one or more mammalian beta defensins comprise an amino acidsequence having at least 80% sequence identity to SEQ ID NO
 2. 12. Themethod of claim 1, wherein the one or more mammalian beta defensinscomprise a variant of human beta defensin 2, wherein no more than 10amino acids have been changed compared to SEQ ID NO
 2. 13. The method ofclaim 1, wherein the one or more mammalian beta defensins are capable ofdownregulating the activity of at least one pro-inflammatory cytokine ina human cell selected from the group consisting of human PBMCs, humanCD14+ monocyte-derived dendritic cells, a human monocyte cell line, andhuman immature dendritic cells.
 14. The method of claim 13, wherein theproinflammatory cytokine is selected from the group consisting of TNFα,IL-23, and IL-1β.
 15. The method of claim 1, wherein the one or moremammalian beta defensins are capable of up-regulating the activity of atleast one anti-inflammatory cytokine in a human cell selected from thegroup consisting of human PBMCs, human CD14+ monocyte-derived dendriticcells, a human monocyte cell line, and human immature dendritic cells.16. The method of claim 15, wherein the anti-inflammatory cytokine isIL-10.
 17. The method of any of claim 1, wherein the one or moremammalian beta defensins are administered at least once daily.
 18. Themethod of any of claim 1, wherein the one or more mammalian betadefensins are administered at a daily dosage of from about 0.0001-10mg/kg.
 19. The method of claim 1, wherein a defensin is administeredorally and a defensin is administered parenterally and the orallyadministered defensin and parenterally administered defensin are thesame.
 20. The method of claim 1, wherein a defensin is administeredorally and a defensin is administered parenterally and the orallyadministered defensin and parenterally administered defensin aredifferent.